Primer group for detecting blood flow infection related pathogens, kit and application
By designing primer sets covering 13 bloodstream infection pathogens and optimizing amplification concentrations, combined with a next-generation sequencing platform, the limitations of detection range and amplification efficiency in existing technologies have been resolved, achieving high sensitivity and high specificity in bloodstream infection detection, suitable for rapid clinical diagnosis.
Patent Information
- Application Number
- CN202511748379.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-26
- Publication Date
- 2026-03-17
AI Technical Summary
Existing technologies are insufficient to simultaneously cover the detection of Gram-negative bacteria, Gram-positive bacteria, and fungi using multiplex PCR. They suffer from limitations in detection range, variations in amplification efficiency, and deviations in detection results, failing to meet the need for rapid and accurate diagnosis of bloodstream infections.
A primer set was designed to cover 13 common bloodstream infection pathogens, including Gram-negative bacteria, Gram-positive bacteria, and fungi. Primer concentration optimization was used to improve amplification uniformity, and combined with a next-generation sequencing platform, high sensitivity and high specificity detection were achieved.
It enables simultaneous detection of 13 pathogens with a detection limit of 10 copies/µL, meeting the needs for early diagnosis of trace infections. The test results are highly accurate, the operation is simple, and it is suitable for clinical application.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of blood stream infection detection, and relates to a primer group for blood stream infection related pathogen detection, a kit and application. BACKGROUND
[0002] Blood stream infection is one of the common critical illnesses in clinic, which has rapid onset, rapid progression and high mortality. Whether the pathogen can be determined early and precise anti-infection treatment is implemented is directly related to the prognosis of the patient. At present, blood culture is still the gold standard for clinically diagnosing blood stream infection, but this method has significant limitations such as long detection period, limited sensitivity, easy to be affected by antibacterial drug pretreatment, and often difficult to meet the rapid diagnosis needs of critical patients. With the development of molecular diagnosis technology, the detection method based on nucleic acid amplification provides a new way for the rapid screening of blood stream infection. Among them, the multiplex PCR technology is concerned due to its high throughput and high efficiency. However, the multiplex PCR primer design in the existing reports focuses on the detection of pathogens of the same genus or the same type, such as only for gram-negative bacteria or several common bacteria, which is difficult to achieve comprehensive coverage across genera and species, especially lacking an effective system for simultaneous detection of gram-positive bacteria and fungi. In addition, there are differences in amplification efficiency of different primers in the multiplex amplification system, which easily leads to missed detection of some targets or signal bias, seriously affecting the accuracy and clinical applicability of the detection results. Although the metagenomic sequencing technology has a wide coverage, its operation is complex, the cost is high, and the human background interference is large, which has not been popularized in routine clinical application. Therefore, developing a primer group capable of simultaneously detecting a plurality of common blood stream infection pathogens including gram-negative bacteria, gram-positive bacteria and fungi, and having high sensitivity, high specificity and good amplification uniformity has become a key problem to be solved in the current clinical testing field. SUMMARY
[0003] The purpose of the present application is to solve the technical problem that blood stream infection related pathogen detection is difficult to simultaneously cover gram-negative bacteria, gram-positive bacteria and fungi.
[0004] To this end, the present application provides a primer group for blood stream infection related pathogen detection, which can realize the detection of 13 pathogens in blood stream infection related pathogens, and these pathogens cover common gram-negative bacteria, gram-positive bacteria and fungi, and have good sensitivity and specificity.
[0005] In one aspect, the present application relates to a primer group for blood stream infection related pathogen detection, which has an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 1-26, and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 27-52.
[0006] In another aspect, the present application relates to a kit for detecting blood stream infection related pathogen, the kit comprising a primer mix, wherein the primer mix comprises the primer set according to claim 1.
[0007] Further, in the kit for detecting blood stream infection related pathogen provided by the present application, the kit comprises the primer mix, a DNA polymerase, dNTPs and water.
[0008] Further, in the kit for detecting blood stream infection related pathogen provided by the present application, the kit further comprises a linker Barcode for sequencing library construction.
[0009] In another aspect, the present application relates to a method for constructing a library of blood stream infection related pathogen, comprising: extracting nucleic acid from a blood sample, performing multiplex PCR amplification and purification on the nucleic acid using the primer set to obtain a pure multiplex PCR product, introducing a linker Barcode for sequencing library construction by PCR amplification of the pure multiplex PCR product, purifying and sequencing the PCR product after adding the linker to obtain a library fragment.
[0010] In another aspect, the present application relates to a method for detecting blood stream infection related pathogen for non-diagnostic purposes, comprising: constructing a library using the method for constructing a library of blood stream infection related pathogen, analyzing data of the obtained library fragment by sequencing, and determining the pathogen in the blood sample.
[0011] Further, in the method for detecting blood stream infection related pathogen for non-diagnostic purposes provided by the present application, the sequencing method is second-generation sequencing.
[0012] Further, in the method for detecting blood stream infection related pathogen for non-diagnostic purposes provided by the present application, the blood stream infection related pathogen is Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter cloacae complex, Streptococcus pneumoniae, Listeria monocytogenes, Haemophilus influenzae, Stenotrophomonas maltophilia and Candida albicans.
[0013] Compared with the prior art, the technical solution provided by the present application has at least the following beneficial effects or advantages: This invention utilizes a primer set design that enables simultaneous detection of 13 of the most common bloodstream infection pathogens in clinical practice, covering three major categories: Gram-negative bacteria, Gram-positive bacteria, and fungi. This overcomes the limitations of existing technologies, which often restrict detection to pathogens of the same genus or class. Through systematic primer concentration optimization, this invention significantly improves the amplification uniformity of each primer pair in the multiplex PCR system, effectively avoiding detection bias caused by differences in amplification efficiency and ensuring the accuracy and reliability of the detection results. The detection method of this invention has extremely high sensitivity, with a detection limit of 10 copies / µL, far superior to traditional blood culture methods, and can meet the clinical diagnostic needs for early, trace infections. Furthermore, the kit and library preparation protocol provided by this invention are easy to operate and perfectly compatible with next-generation sequencing platforms, making them particularly suitable for clinical application and providing an efficient technical means for the rapid and accurate diagnosis of bloodstream infections. Detailed Implementation
[0014] The technical solution of the present invention will be described below with reference to embodiments; however, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentage content. Unless otherwise specified, all ratios in the following embodiments refer to mass ratios.
[0015] Example 1 This embodiment provides the design and validation of primer sets.
[0016] Based on statistical results from clinical samples of bloodstream infections in hospitals, 13 major pathogens involved in bloodstream infections were selected, covering Gram-negative bacteria, Gram-positive bacteria, and fungi. Primer design is particularly crucial in the use of multiplex PCR combined with next-generation sequencing. The designed primers not only need to have specific pathogen recognition capabilities but also need to introduce barcodes into the amplified fragments for subsequent identification and segmentation of sequencing data, while ensuring that the fragment length after library construction is approximately 250-300 bp. Furthermore, all primers should operate under identical reaction conditions. However, due to differences in amplification efficiency among different primers at the same concentration, the accuracy of sequencing results and pathogen identification may be affected. Therefore, it is necessary to optimize primer concentrations to reduce the bias in genome amplification within the same reaction system.
[0017] The primer sequences designed for the detection of bloodstream infection-associated pathogens are shown in Table 1.
[0018] Table 1: Primer sequences
[0019] To test the sensitivity of each primer pair, the following steps were performed: Step 1: Template preparation and amplification. Genomic DNA was extracted from 13 standard strains of bloodstream infection-associated pathogens. PCR amplification was performed using a PCR amplification system consisting of 15 μL Green Taq Mix, 1 μL upstream primer (10 μM), 1 μL upstream primer (10 μM), 1 μL DNA template (150 ng / μL), and 12 μL ddH2O. The amplification program was 94℃ for 4 min, (94℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min) / 30 cycles, and 72℃ for 5 min. Two sets of corresponding primers were used for each strain. Step 2: Product detection. PCR products are analyzed using 1% agarose gel electrophoresis, and the banding is observed. Step 3: Result interpretation. Primers that can amplify a single target band are considered to have good sensitivity; otherwise, primers need to be redesigned and the above verification process needs to be repeated.
[0020] PCR validation showed that all 26 primer pairs exhibited good sensitivity, with PCR products appearing as a single bright band of approximately 150 bp in 1% agarose gel electrophoresis. However, differences in band brightness reflected variations in amplification efficiency between primers, necessitating further concentration optimization to improve amplification uniformity.
[0021] To verify the specificity of each primer pair, the following steps were performed: Step 1: Genomic DNA from HEK-293T cells derived from human embryonic kidney cells was extracted using a cell genomic extraction kit as a human background. Step 2: Mix the genomic DNA of the remaining 11 bloodstream infection-related pathogen standard strains (excluding the microorganisms corresponding to the test primer pairs) and the genomic DNA of HEK-293T cells as DNA templates (10 ng / μL), and perform PCR amplification according to the PCR amplification system and PCR amplification procedure in the sensitivity test method. Step 3: Product detection. PCR products are analyzed using 1% agarose gel electrophoresis, and the banding is observed. Step 4: Result interpretation. Primers that cannot amplify bands are considered to have good specificity; otherwise, primers need to be redesigned and the above verification process needs to be repeated.
[0022] PCR validation showed that all 26 primer pairs exhibited good specificity, and no non-specific bands appeared in the absence of target genomic DNA, confirming that the designed 26 primer pairs targeting the 13 target pathogen strains had good specificity.
[0023] To ensure the uniformity of each primer pair, follow these steps: Genomic DNA from 13 standard strains of bloodstream infection-associated pathogens was used as templates. PCR amplification was performed according to the PCR amplification system and procedure in the sensitivity testing method to obtain 26 PCR products for each primer pair. The genomic DNA from HEK-293T cells was then mixed with the 26 purified DNA fragments, with a final concentration of 10 for each fragment. 5 Copies / µL were used as templates, and multiplex PCR amplification was performed according to the PCR amplification system and program in the sensitivity test method. The multiplex PCR amplification system consisted of 4μL Primer mix, 3μL DNA, 13μL ddH2O, and 20μL mPCR Taq enzyme (2×). The multiplex PCR amplification program was 95℃ for 3 min, (95℃ for 10 s, 60℃ for 30 s, 72℃ for 1 min) / 30 cycles, and 72℃ for 5 min. The multiplex PCR products were used to construct libraries according to the library construction kit instructions, and second-generation sequencing and data analysis were performed. Based on the sequencing results, the concentration of each primer pair in the mixed primers was adjusted according to the formula uniformity = (target primer reads / average of total sequencing reads). The adjustment results are shown in Table 2.
[0024] Table 2: Optimization results of each primer pair in the primer set
[0025] In Table 2, the uniformity of amplification results was achieved by adjusting the concentration of each primer pair to increase or decrease the primer amplification efficiency, thus obtaining the final Primer mix.
[0026] Example 2 This embodiment provides validation results for bloodstream infection simulation samples and clinical samples.
[0027] Following the method in Example 1, 10 DNA fragments related to Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Candida albicans were selected from the 26 pathogen DNA fragments obtained and mixed, with a final concentration of 10 for each DNA fragment. 8 copies / µL, then serially diluted to 10 6 copies / µL, 10 5 copies / µL, 10 4 copies / µL, 10 3 copies / µL, 10 2 copies / µL, 10 1 copies / µL, 10 0Using copies / µL and 0 copies / µL as templates, multiplex PCR amplification was performed according to the multiplex PCR amplification system and program in Example 1. The resulting multiplex PCR products were purified and introduced into the corresponding Illumina sequencing adapter barcode for further PCR amplification and purification. The resulting products were the constructed DNA library. The concentration of the constructed library fragments was measured using a Qubit 4 fluorometer, and then second-generation sequencing and data analysis were performed using the Illumina HiSeq 2500 platform. The copy number of reads greater than or equal to 100 was defined as the detection limit of this method.
[0028] The logarithm of the reads from the sequencing results of the 24 primer pairs is shown in Table 3.
[0029] Table 3: Validation results of simulated bloodstream infection samples
[0030] As shown in Table 3, with sequencing reads ≥ 10 2 The detection limit of this method is defined as copies. Therefore, the detection limit of the primer set provided by this invention is 10 copies / µL, which has good application effect.
[0031] Genomic DNA was extracted from blood samples using the MolPure® Magnetic Circulating Cell-Free DNA Kit according to the manufacturer's instructions. Multiplex PCR amplification was performed using a mixture of 4 μL Primer mix (primer pair concentrations refer to Table 2), 10 μL blood sample, 6 μL ddH2O, and 20 μL mPCR Taq enzyme (2×), following the multiplex PCR amplification procedure described in Example 1. The resulting multiplex PCR products were purified, introduced into Illumina sequencing adapters, and then subjected to further PCR amplification and purification. The resulting product constituted the constructed DNA library. (With ≥10 reads...) 2 Copies are defined as a positive test result, 10 6 DNA fragments at a concentration of copies / µL served as a positive control, with blood culture results used as a comparison. The experimental results are shown in Table 4.
[0032] Table 4: Clinical Sample Validation Results of Bloodstream Infection
[0033] As shown in Table 4, the method provided by this invention has a specificity of 85.71%, a sensitivity of 88.37%, an effectiveness of 88.00%, a positive predictive value of 97.44%, and a negative predictive value of 54.55% in clinical samples. Among them, the five false negative samples did not belong to the 13 pathogens covered by this application. Using DNA fragments as positive controls can effectively avoid the false positive and false negative problems in the conventional identification method detection process, making the detection results more reliable.
[0034] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.
Claims
1. A primer set for blood stream infection-related pathogen detection, characterized by, The primer set has an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 1-26, and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 27-52.
2. A kit for the detection of blood stream infection associated pathogen, characterized in that, The kit comprises a Primer mix comprising the primer set of claim 1.
3. The kit for detection of blood stream infection related pathogen according to claim 2, characterized in that, The kit comprises the Primer mix, a DNA polymerase, dNTPs and water.
4. The kit for detection of blood stream infection related pathogen according to claim 3, characterized in that, The kit further comprises a linker Barcode for sequencing library construction.
5. A method of constructing a library of blood stream infection associated pathogens, characterized by, It comprises: extracting nucleic acids from a blood sample, performing multiplex PCR amplification and purification on the nucleic acids using the primer set of claim 1 to obtain a pure multiplex PCR product, introducing a linker Barcode for sequencing library construction by PCR amplifying the pure multiplex PCR product, purifying and sequencing the PCR product after adding the linker to obtain library fragments.
6. A method of detecting a pathogen associated with bloodstream infection for non-diagnostic purposes, characterized in that, It comprises: performing library construction using the library construction method of blood stream infection related pathogens of claim 5, analyzing the obtained library fragments, and determining the pathogens in the blood sample.
7. The method for detecting a blood infection-related pathogen for non-diagnostic purposes according to claim 6, characterized in that, The sequencing method is second-generation sequencing.
8. The method of claim 6, wherein the method is for detecting a pathogen associated with bloodstream infection for non-diagnostic purposes. The blood stream infection related pathogens are Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter cloacae complex, Streptococcus pneumoniae, Listeria monocytogenes, Haemophilus influenzae, Stenotrophomonas maltophilia and Candida albicans.